GENETIC VARIATION IN SYMPATRIC AND ALLOPATRIC POPULATIONS OF HYBRIDIZING FRESHWATER SNAIL SPECIES(VIVIPARUS ATER AND V. CONTECTUS)

GENETIC VARIATION IN SYMPATRIC AND ALLOPATRIC

POPULATIONS OF HYBRIDIZING FRESHWATER SNAIL

SPECIES (VIVIPARUS ATER AND V. CONTECTUS)

MASAYA KATOH

1

and GEORG RIBI

2

'Seikai National Fisheries Research Institute, Fukai-Ohta 148-446, Ishigaki 907-04, Japan. 2Zoologisches Museum der Universitat ZUnch, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

(Received 21 June 1996; accepted 31 January 1997)

ABSTRACT

To estimate the geographical extent of introgression, we studied the genetic structure of sympatric and allopatric populations of hybridizing freshwater snail species Viviparus ater and V. contectus in central Europe. Six allozyme loci which were variable in Lake Garda, Italy in a previous study (five nearly diagnostic loci between the two species and one highly poly-morphic locus in V. contectus) were analyzed from ten sympatric locations and four allopatric populations each for the two species. Presumably introgressed genes (low allele frequencies) were found from at least one locus in seven out of the ten sympatric sites. These seven sites covered most of northern Italy. The data indicate that introgression has occurred from Viviparus contectus to V. ater and vice versa. There-fore, there is a possibility of widespread introgression or mosaic zones in nature. However, we cannot rule out that the observed patterns are due to the shared ancestry. V. ater possessed low genetic variation (the jackknifed mean of Wright's F^ ± S.E. over four loci was 0.041 ± 0.004). On the other hand, V. contectus showed high genetic differentiation (the jackknifed mean of Fgr ± S.E. over six loci was 0.546 ± 0.166). Although introgression may have caused evolution-ary changes in V. ater and V. contectus, it was not strong enough to level out the genetic differences between the two species, which may have originated from isolation among populations in V. contectus and a past bottleneck event in V. ater.

INTRODUCTION

Hybridization and introgression are at the

centre of evolutionary interest because they

can help to understand how reproductive

isola-tion evolves. Examples of hybridizaisola-tion and

introgression have been reported from many

plant (Rieseberg & Wendel, 1993) and animal

Address for correspondence: Dr. M Katoh, Ishigaki Tropical Station. Seikai National Fisheries Research Institute, Fukoi-Ohta \AS-M6, Ishi-gaki, Okinawa 907-04, Japan. +81-9806-8-2573 (fax) mkatoh®jnf-ilsjfTrc.go.jp (e-mail)

species (Harrison, 1993) including molluscs

(Woodruff & Gould, 1987; McDonald, Seed &

Koehn, 1991; Falniowski, Kozik & Szarowska,

1993; Porter & Ribi, 1994; Katoh & Ribi, 1996;

Yokogawa, 1996). Natural hybridization takes

place in narrow area of contact zones or mosaic

zones (Rand & Harrison, 1989). Through

introgression, species may acquire genetic

polymorphism (Echelle & Connor, 1989).

Hybridization and introgression between

species may affect evolutionary events

includ-ing speciation and phylogenetic relationships.

First generation hybrids between the

fresh-water snails Viviparus ater and V. contectus

were collected in nature and their hybrid status

confirmed with allozyme markers (Katoh &

Ribi, 19%). The estimated frequency of the F,

hybrids was 0.74% in Lake Garda, Italy (Katoh

& Ribi, 19%). Allozyme data from Lake Garda

are consistent with the hypothesis of gene

introgression from V. ater into V. contectus

(Porter & Ribi, 1994) and vice versa (Katoh &

Ribi, 19%). Crossing experiments revealed a

Mendelian hybrid system with one pair of

linked loci (GPI and PNP; Katoh & Ribi,

19%). V. ater and V. contectus co-occur at high

densities in man-made structures such as

har-bours and canals. Their dispersal is limited

because of brooding. The purpose of this study

is to estimate the extent of introgression of

allozymes based on allele frequency data.

MATERIALS AND METHODS A total of 890 freshwater snails was collected from ten sympatric locations and four allopatric popula-tions each, of Viviparus ater (499 snails) and V. con-tectus (391 snails) in Italy, Switzerland, and Germany between October 1992 and August 1994 (Fig. 1 and Table 1). Each allopatric population was coded by a combination of a number which represents a specific site and a lower case letter which indicates an

exist-MASAYA KATOH & GEORG RIBI ing species. Sympatric sites were coded by a number

only. If both species were present at a sampling site or in a lake where a sampling site is located, we treated the site as sympatric in this study. Only V. ater was collected at the sympatric site Magadino, Lake Maggiore. Lake Chiem, Germany (Population lc) belongs to the Danube drainage system. Lake Constance (2a) is part of the Rhine system. The remaining sites are in the Po plain. Snails were trans-ported alive to the University of Zuurich-Irchel, Switzerland and frozen at -75°C until analyzed electrophoretically.

Standard horizontal starch-gel electrophoresis was performed. Procedures for tissue-extract preparation, and electrophoresis were similar to those of Murphy, Sites, Buth & Haufler (1990) with minor modifica-tions. Five allozyme loci were nearly diagnostic between the two species in Lake Garda, Italy in the previous study (Katoh & Ribi, 1996). One locus [PGDH] was highly polymorphic in V. conleclus. These six allozyme loci (fi-galactosidase, EC 3.2.1.22, fSGAL; glucose-6-phosphate isomerase, EC 5.3.1.9,

GPI; malate dehydrogenase-1, EC 1.1.1.37, MDH-1; phosphoglucomutase, EC 5.4.2.2, PGM; phosphoglu-conate dehydrogenase, EC 1.1.1.44, PGDH; purine-nucleoside phosphorylase, EC 2.4.2.1, PNP) were stained for the newly collected 890 snails. We used a single gel buffer system of 'JRP' following Avise, Smith & Ayala (1975) for the six allozyme loci studied. Alleles which were most abundant at each locus of V. ater in Lake Garda were designated 100, and the other alleles were named according to their relative mobilities. /•'-statistics (Weir & Cockerham, 1984) were calculated for each locus to determine genetic structure among collections within species. The method by Weir & Cockerham (1984) gives an unbiased estimate of Wright's (1978) F^.

RESULTS

Genes presumed to be introgressed were found

at one or two loci in seven out of ten sympatric

Figure 1. Collection sites for sympatric and allopatric populations of Viviparus ater and Viviparus contectiis in

507

Table 1. Sampling sites and dates for sympatric and allopatric populations of Viviparus ater and

Viviparus contectus in central Europe.

Code' 1c 2a 3 4a 5a 6a 7 8 9 10 11 12 13 14 15c 16c 17 18c Location

Lake Chiem, Germany

Horn, Lake Constance, Switzerland Magadino, Lake Maggiore, Switzerland Bissone, Lake Lugano, Italy

Porlezza, Lake Lugano, Italy Lake Piano, Italy

Gravedona, Lake Como, Italy Campo, Lake Como, Italy Cernobbio, Lake Como, Italy Sarnico, Lake Iseo, Italy

Lake Garda, Italy from Katoh & Ribi (1996) East of G6ito, Italy

South of G6ito, Italy Noventa, Italy Rosolina, Italy Stretti 1, Italy Stretti 2, Italy

Al Ponte, Grado-Fiumicello, Italy

Collection date 28/9/93 10/8/94 7/8/94 29/10/92 29/10/92 29/10/92 7/5/94 6/5/94 9/5/94 1 & 22/5/93 2/5/93 2/5/93 23/10/92 25/10/92 20/3/93 21/3/93 22/3/93 Collected species V. conectus V. ater V. ater V. ater V. ater V. ater

V. ater & V. contectus V. ater Si V. contectus V. ater&t V. contectus V. ater & V. contectus V. ater & V. contectus V. ater Si V. contectus V. ater & V. contectus V. ater & V. contectus V. contectus

V. contectus

V. ater &. V, contectus V. contectus

'a and c indicate the existing species Viviparus ater and Viviparus contectus, respectively, at the allopatric sites.

Table 2. Allele frequencies of allopatric and sympatric populations of Viviparus aferfrom Switzerland and Italy. See Table 1 for names of the population sites.

Locus/ allele 2a 3 4a 5a 6a Populations 7 8 9 10 11 12 13 14 17 PGAL '105 '100 •94 GPI •119 •100 MDH-1 •143 '100 *44 PGM '100 *93 *82 PGDH '148 '100 PNP '100 N - - - £LQ5 -0.99 1.00 1.00 1.00 1.00 1.00 0.95 1.00 1.00 -0.998 1 0.01 _ - - _ _ _ - - 0.000 - - - 0.Q07 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.993 1 - - - 0.01 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 0.99 -1.00 -1.00 0.997 1 - - - 0.003 1.00 1.00 0.93 0.92 1.00 1.00 0.99 1.00 1.00 0.998 1 - - - 0.002

0.07 0.08 - - M l - - 0.000

0.01 - - - 0.0Q2 1.00 1.00 0.99 1.00 1.00 1.00 1.00 1.00 1.00 0.998 1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.000 1 80 34 58 60 15 45 40 40 42 293 .00 .00 .00 .00 .00 .00 5 1.00 1.00 1.00 0.96 0.04 1.00 1.00 28 0,02 0.86 0.05 1.00 1.00 1.00 1.00 1.00 11 1.00 1.00 1.00 0.99 0.01 1.00 1.00 41 Frequencies of proposed introgressed alleles are underlined.

The most common allele in V. ater is designated as '100; "-" means 0.00. Data for Population 11 (Lake Garda, Italy) are from Katoh & Ribi (1996).

Table 3. Allele frequencies of allopatric and sympatric populations of Viviparus contectus from Germany and Italy. See Table 1 for names of the population sites.

Locus/ allele 1c 10 Populations 11 12 13 14 15c 16c 17 18c PGAL •105 •100 •94 GPI •144 •119 •100 MDH-1 •100 •84 •44 PGM '100 '93 •82 •78 PGDH •148 •100 PNP •122 '100 N 1.00 -_ 1.00 -1.00 -0.10 0.90 _ 1.00 _ -1.00 5 0.99 -0.01 _ -1.00 0.01 -0.99 -_ 1.00 _ _ 1.00 -1.00 39 1.00 -1.00 -— 1.00 -0.09 0.91 _ _ 1.00 -1.00 35 0.89 QA1 — _ -1.00 -1.00 -0.09 0.91 _ _ 1.00 -1.00 40 1.00 -— — 1.00 -— 1.00 0.06 0.10 0.84 _ 0.65 0.35 0.53 0.47 35 0.995 0.005 0.000 0.000 0.944 0.056 OJJOS 0.001 0.991 PJJ22 0.031 0.947 0.000 0.470 0.530 0.974 0JJ26 383 1.00 -1.00 -_ 1.00 -— 1.00 _ 1.00 _ 0.31 0.69 16 1.00 -1.00 -_ 1.00 -0.11 0.89 _ 1.00 _ 0.37 0.63 31 1.00 -1.00 -1.00 -0.40 0.60 _ 0.85 0.15 0.90 0.10 10 1.00 -0.02 0.98 -1.00 -0.74 0.26 _ 0.62 0.38 -1.00 21 1.00 -1.00 -1.00 -0.60 0.40 _ 0.33 0.67 0.24 0.76 68 1.00 -0.99 0.01 -1.00 -0.54 0.45 0.01 0.50 0.50 0.17 0.83 60 1.00 -1.00 -1.00 -0.61 0.39 _ 0.05 0.95 0.02 0.98 31 Frequencies of proposed introgressed alleles are underlined.

The most common allele in V. ater is designated as '700; "-" means 0.00. Data for Population 11 (Lake Garda, Italy) are from Katoh & Ribi (1996).

sites (Viviparus ater in Table 2 [Populations 8,

13, 14 and 17; V. contectus in Table 3

[Popula-tions 7,9,10 and 17]). These seven sites covered

most of northern Italy. Inferred directions of

introgresson were from V. contectus to V. ater

(Table 2) and vice versa (Table 3). However,

two allopatric populations (Table 2;

Popula-tions 4a and 5a) of V. ater in Lake Lugano

pos-sessed PGM*82 and PGDH*148, which were

rather common alleles in V. contectus (Table 3),

at low frequencies.

Viviparus ater had low mean heterozygosity

(overall mean H,. ± S.D. = 1.1 ± 1.4%) and

little genetic differentiation (Table 2). The

jackknifed mean of Wright's F^ ± S.E. over

four loci was low (0.041 ± 0.004), which also

indicates genetic homogeneity (Table 4). On

the other hand, V. contectus showed high mean

expected heterozygosity (overall mean H

c

±

S.D. = 11.5 ± 7.6%) and high genetic

differ-entiation especially at four loci (GPI, PGM,

PGDH, and PNP, Table 3). The jackknifed

mean of F^ ± S.E. over six loci was very high

(0.546 ± 0.166), which indicates high genetic

differentiation among populations (Table 4).

Sympatric populations of V. contectus in Lake

Como, Italy (populations 7, 8 and 9) possessed

only GPI*100 and PNP*100 at one pair of the

linked loci (Table 3). GPI at the other

sym-patric and allosym-patric populations were nearly

substituted by *119 (Table 3). Frequencies of

PNP*100, which was a common allele in V. ater

and only 2.6% in V. contectus in Lake Garda,

were more than 50% in most populations of V.

contectus in this study.

DISCUSSION

Presumably introgressed alleles were found

from most sympatric populations of Viviparus

ater and V. contectus in northern Italy. Some

allopatric populations had genes which may

have had their origin in introgression. But

be-cause shared alleles between species can be

due to mutation or to common ancestry, the

Table 4. Hierarchical F-statistics for Viviparus ater and Viviparus contectus with jackknifed means and standard errors (S.E.).

Locus Viviparus ater PGAL MDH-1 PGDH PGM Mean S.E. Viviparus contectus PGAL GPI MDH-1 PGDH PGM PNP Mean S.E. FG 0.476 0.001 0.005 0.042 0.063 0.213 -0.099 -0.001 0.003 -0.134 0.115 0.080 0.029 0.084 0.046 -0.001 -0.005 0.040 0.041 0.004 0.082 0.989 -0.003 0.492 0.329 0.296 0.546 0.166

origin of each allele cannot be inferred from

allele frequency data (Harrison, 1990).

There-fore, we cannot rule out that observed patterns

are due to shared ancestry. In fact, although

PNP*100 in V. contectus from Lake Garda was

interpreted as introgressed allele in Katoh &

Ribi (19%), the PNP*100 in Lake Garda may

be a simple polymorphic allele because PNP

was highly variable at other populations in this

study. However, V. ater and V. contectus can

hybridize in nature and frequency of F] hybrids

was 0.74% in Lake Garda (Katoh & Ribi,

19%). F! hybrids are viable and mainly males

(Trllb, 1990; Katoh & Ribi, 19%). Females

crossed with male F, hybrids produced

back-crossed offspring (Triib, 1990). Therefore,

there is a possibility of widespread

intro-gression or mosaic zones in nature. Moreover,

the alleles PGM*82 and PGDH*148 of V. ater

in Lake Lugano (allopatric sites) may have

introgressed in the past when the two species

coexisted.

Even if hybridization and introgression

occur at many places, we do not expect that

frequencies of introgressed alleles increase in

time because Viviparus ater and V. contectus

have reduced fitness of F, hybrids. In

Vivi-parus, crosses between V. ater females and V.

contectus males produced 50% of offspring of

normal intraspecific V. ater fecundity (Trilb,

1990). The reverse cross produced only 2% of

intraspecific V. contectus fecundity. The

fecun-dity of the back-crossed snails was about half of

normal conspecific matings. In spite of these

disadvantages, these species could maintain a

low level of gene leakage through interspecific

and back-cross matings.

The six allozyme loci indicated high genetic

differentiation in Viviparus contectus and low

genetic differentiation in Viviparus ater. The

presence of many isolated small populations

seems to cause high differentiation in V.

con-tectus. V. contectus prefers small streams and

swamps where populations tend to be small

and V. ater lives primarily in larger bodies of

water such as lakes and rivers where

popula-tions are large (Franz, 1932; personal

observa-tion). Other brooding Viviparus species in the

United States show large genetic differentiation

among and within drainage systems (Katoh

& Foltz, 1994). In general, freshwater

gastro-pod populations tend to be genetically

differ-entiated (Brown & Richardson, 1988; Jarne &

Delay, 1991). In addition, the low genetic

varia-tion found in V. ater based on 13 allozyme loci

suggests a severe bottleneck event in the past

(Porter & Ribi, 1994). Although low

introgres-sion may have happened several times at many

places where V. ater and V. contectus coexisted,

the genetic differences which were observed

between V. ater and V. contectus in this study

can be explained by the presence of many

isolated small populations of V. contectus and a

past bottleneck event in V. ater.

ACKNOWLEDGEMENTS

We thank R.T. Dillon and B.R. Anholt for helpful comments on the manuscript. D.W. Foltz (LSU) pro-vided us his computer program for genetic analysis. We also thank D. Furrer for running gels and P. Brauchli for drawing a map. This work was supported by Swiss NSF grants 25310.88 and 31-36363.92 to G.R.

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